The greatest challenge of the post genomic era is to uncover functions of every individual human gene. Development of novel high throughput functional genomics tools are critical for genome-wide discovery and validation of drug targets, and to open avenues for newer concepts in drug therapy and diagnostics of complex human diseases. The recent introduction of gene knockdown technology based on small interfering RNAs (siRNA) holds a promise to revolutionize gene functional analysis. The goal of the proposed program is to develop and make commercially available a comprehensive set of genome-wide siRNA libraries in lentiviral vectors that are more effective and cost-efficient than the currently available collection of synthetic siRNAs. Under Phase I funding, the biosafe feline immunodeficiency virus (FIV) lentiviral siRNA cloning vectors and technology for construction of high complexity siRNA libraries were developed. The Human 1.5K siRNA library, comprising of a redundant set of 7500 siRNAs for 1500 genes, was constructed and functionally tested in the radiation resistance HT1080 cell model. High-throughput technology for genetic screens of radiation-resistance genes, based on a combination of siRNA libraries and Affymerix GeneChip(r) microarrays, was developed. Lentiviral siRNA cloning vectors were released as commercial products and ready-to-use, prepackaged in viral particles, human 1.5K and 8.5K siRNA libraries are currently in the product launch cycle. Under Phase II funding, we propose to extend the program towards development and commercialization of a comprehensive set of genome-wide 50K human, 40K mouse and 30K rat siRNA libraries in lentiviral vectors with a selection of different reporters and tet-regulated expression of siRNAs. siRNA libraries will allow the performance of global searches for key genes involved in a variety of diseases and to validate new drug targets by arbitrary selective inactivation of diseaseassociated genes in the context of the living cells. The established technology will be applied, in collaboration with the Cleveland Clinic Foundation, for identification of targets for drug development, aiming at prostate cancer. The anticipated outcomes of the proposed research and product development program will be a commercially available set of kits comprising of lentiviral vectors and siRNA libraries for high-throughput gene functional analysis, custom genomewide functional screen service and validated anticancer drug targets for treatment of prostate cancer.